This invention relates to the detection of nucleic acids. More particularly, this invention relates to the detection and quantitation of charged nucleic acid sequences present in biological fluids.
Detection of specific nucleic acid sequences present in cells is generally known in the art. Southern (J. Mol. Bio. (1975) 98: 503-517) teaches the detection of specific sequences among DNA fragments separated by gel electrophoresis using "blotting" or transfer of the DNA fragments to a membrane, followed by hybridization of denatured DNA fragments with a radioactive probe and autoradiography. This procedure has also been extended to the detection of RNA molecules extracted from cells or tissues. More recently, faster and quantitative "dot-blotting" procedures have been developed for rapid detection of DNA or RNA from tissues or cells.
Recently, considerable interest has been generated in the development of synthetic oligonucleotides as therapeutic or gene expression modulating agents in the so-called antisense approach. These agents, called antisense oligonucleotides, bind to a target single-stranded nucleic acid molecule according to the Watson-Crick or the Hoogstein rule of base pairing, and in doing so, disrupt the function of the target by one of several mechanisms: by preventing the binding of factors required for normal translation or transcription; in the case of an mRNA target, by triggering the enzymatic destruction of the message by RNase H; or by destroying the target via reactive groups attached directly to the antisense oligonuceotide.
Antisense oligodeoxynucleotides have been designed to specifically inhibit the expression of HIV-1 and other viruses (see, e.g., Agrawal (1992) Trends in Biotechnology10:152-158; Agrawal et al. in Gene Regulation: Biology of Antisense RNA and DNA (Erickson and Izant, eds.) Raven Press Ltd., New York (1992) pp. 273-283); Matsukura et al. in Prospects for Antisense Nucleic Acid Therapy of Cancer and AIDS, Wiley-Liss, Inc. (1992) pp. 159-178; and Agrawal (1991) in Prospects for Antisense Nucleic Acid Therapy for Cancer and AIDS, (Wickstrom, ed. ) Liss, New York, pp. 145-148). For example, it has been shown that antisense oligonucleotides having sequences complementary to portions of genomic HIV-1 ribonucleic acid (RNA) inhibit viral replication in early infected cells (Zamecnik et al. (1986) Proc. Natl. Acad. Sci. (USA) 83:4143-4147; Goodchild et al. (1988) Proc. Natl. Acad. Sci (USA) 85:5507-5511).
To further improve their characteristics as antisense agents, chemically modified, nuclease-resistant analogs have been developed which are effective in inhibiting HIV-1 replication in tissue cultures (Sarin et al. (1988) Proc. NatlAcad. Sci. (USA) 85:7448-7451: Agrawal et al. (1988) Proc. Natl. Acad. Sci. (USA) 85:7079-7083; Matsukura et al. (1988) Gene 72:343-347). These analogs include oligonucleotides with nuclease-resistant phosphorothioate internucleotide linkages shown to inhibit HIV-1 replication in both acute infection (Agrawal et al. (1989) Proc. Natl. Acad. Sci (USA) 86:7790-7794) and in chronically infected cell lines (Agrawal et al. (1991 ) in Gene Regulation: Biology of Antisense RNA, (Erickson et al., eds.) Raven Press, New York, pp. 273-284; Vickers et al. (1991) Nucleic Acids Res. 19:3359-3368; Matsukura et al. (1989) Proc. Natl. Acad. Sci. (USA) 86:4244-4248; Agrawal et al. (1988) Proc. NatlAcad. Sci. (USA) 85:7079-7083).
For an antisense therapeutic approach to be effective, oligonucleotides must be introduced into a subject and must reach the specific tissues to be treated. Consequently, there is a need to be able to detect oligonucleotides in body fluids or tissues.
Temsamani et al., Anal. Biochem. 215:54-58(1993) developed a method of extracting oligonucleotides which had been proteolytically digested from body fluid or tissue samples. Total nucleic acids are precipitated from the extracted samples and transferred to a hybridization membrane where they are hybridized under specific conditions to a labelled oligonucleotide that is complementary to the oligonucleotide that was administered to the subject. Presence of the hybridized, labelled oligonucleotide is then detected by standard procedures.
Radiolabelled oligonucleotides have been administered to animals and their distribution within body fluids and tissues has been assessed by extraction of the oligonucleotides followed by autoradiography (see Agrawal et al. (1991) Proc. Natl. Acad. Sci. (USA) 88:7595-7599). As a practical matter, however, these methods are not feasible for use in human patients.
Unfortunately, the various techniques for detecting specific unlabelled nucleic acid sequences present in body fluids or tissues has thus far only been extended to polynucleotides such as large DNA or RNA molecules. Due to the small size of antisense oligonucleotides, special problems relating to nonspecific binding or background, as well as to absence of binding, nondetection, or false negatives exist. Thus, there remains a need to develop procedures for the detection of specific synthetic oligonucleotide sequences present in biological fluids such as body fluids and tissues.